Of great importance to man is the control of pathological cellular proliferation. While certain methods and chemical compositions have been developed which aid in inhibiting, remitting, or controlling cellular proliferation, new methods and compositions are needed.
In searching for new biologically active compounds, it has been found that some natural products and organisms are potential sources for chemical molecules having useful biological activity of great diversity. For example, the diterpene commonly known as taxol, isolated from several species of yew trees, is a mitotic spindle poison that stabilizes microtubules and inhibits their depolymerization to free tubulin (Fuchs, D. A., R. K. Johnson (1978) Cancer Treat. Rep. 62:1219-1222; Schiff, P. B., J. Fant, S. B. Horwitz (1979) Nature (London) 22:665-667). Taxol is also known to have antitumor activity and has undergone a number of clinical trials which have shown it to be effective in the treatment of a wide range of cancers (Rowinski, E. K., R. C. Donehower (1995) N. Engl. J. Med. 332:1004-1014). See also, e.g., U.S. Pat. Nos. 5,157,049; 4,960,790; and 4,206,221.
Marine sponges have also proven to be a source of biologically active chemical molecules. A number of publications disclose organic compounds derived from marine sponges including Scheuer, P. J. (ed.) Marine Natural Products, Chemical and Biological Perspectives, Academic Press, New York, 1978-1983, Vol. I-V; Uemura, D., K. Takahashi, T. Yamamoto, C. Katayama, J. Tanaka, Y. Okumura, Y. Hirata (1985) J. Am. Chem. Soc. 107:4796-4798; Minale, L. et al. (1976) Fortschr. Chem. org. Naturst. 33:1-72; Faulkner, D. J. (1998) Natural Products Reports 15:113-158; Gunasekera, S. P., M. Gunasekera, R. E. Longley and G. K. Schulte (1990) J. Org. Chem., 55:4912-4915.
A prime target for the discovery and design of novel therapeutic agents against cancer is the mitotic apparatus of the cell and more specifically, microtubule assembly and its function (Wilson, L. (1975) xe2x80x9cMicrotubules as drug receptors: pharmacological properties of microtubule proteinxe2x80x9d Ann. N.Y. Acad. Sci. 253:213-231). Ancillary functions of microtubules, including intracellular transport, signal transduction and the maintenance of cellular shape and motility are important factors which contribute to the overall growth of tumor cells and resulting metastases (Dustin, P. (1980) Sci. Am. 243:66-76). Taxol is a microtubule interactive agent whose mechanism of action includes the premature polymerization of tubulin, resulting in hyperstable microtubule formation, blockage of cellular proliferation in the G2/M phase of the cell cycle, mitotic spindle disorganization and cell death. Additional compounds, which are chemically unrelated to Taxol, are rapidly coming onto the scene which share a similar mechanism of action with Taxol and are the subject of intense research into their potential as novel antitumor agents. These include the epothilones A and B, macrolides isolated from a myxobacterium, Sorangium cellulosum (Bollag, D. M., P. A. McQueney, J. Zhu et al. (1995) Cancer Res. 55:2325-2333); eleutherobin, obtained from a marine soft coral (Lindel, T., P. R. Jensen, W. Fenical et al. (1997) J Am. Chem. Soc. 119:8744-8745); laulimalide, isolated from a marine sponge (Mooberry, S. L., G. Tien, A. H. Hernandez et al. (1999) Cancer Res. 59:653-660); and discodermolide isolated from a marine sponge (Gunasekera, S. P., M. Gunasekera, R. E. Longley (1990) J. Org. Chem. 55:4912-4915 and Ter Haar E., R. J. Kowalski, E.Hamel, et. al. (1996) Biochemistry 3:243-250). All of these compounds induce microtubule hyperstabilizing activity and are cytotoxic in vitro to tumor cells in the nanomolar range.
The success of chemotherapy for the treatment of various cancers can be substantially negated though cellular mechanisms which have evolved to enable neoplastic cells to subvert the cytotoxic effects of the drug. Some cells have developed mechanisms, which confer resistance to a number of structurally unrelated drugs. This multi-drug resistance (or MDR) phenomenon may arise through a number of different mechanisms. One of these involves the ability of a cell to reduce intracellular concentrations of a given drug through efflux from cytoplasm through and out the cell membrane by a series of unique ATP-dependent transporter proteins called-P-glycoproteins (Pgp) (Casazza, A. M. and C. R. Fairchild (1996) Cancer Treat Res. 87:149-171). The surface membrane, 170 kDa Pgp, is encoded by the mdr-1 gene and appears to require substrate binding before transport begins. A wide range of compounds including a number of structurally unrelated chemotherapeutic agents (adriamycin, vinblastine, colchicine, etoposide and Taxol), are capable of being transported by Pgp and render the cell resistant to the cytotoxic effects of these compounds. While many normal cell types possess Pgp, in general, tumor cell lines, which possess high levels of mRNA specific for Pgp, also exhibit overexpression of membrane Pgp and demonstrate resistance to various drugs. This intrinsic resistance can be increased multifold by incubation of cells with stepwise increasing doses of a particular drug over a period of several months. This can be further facilitated by the addition of the MDR reversal agent, verapamil (Casazza, A. M. and C. R. Fairchild (1996) supra) in combination with the particular drug. Drug resistant cell lines produced in this fashion exhibit resistance to drug cytotoxicity from 20 to 500 fold, compared to parental cell lines.
An additional target for cancer drug discovery is a high molecular weight membrane protein associated with multi-drug resistance properties of certain tumor cells known as the multidrug resistance-associated protein (MRP). MRP is a 190 kD membrane-bound glycoprotein (Bellamy, W. T. (1996), Annu. Rev. Pharmacol. Toxicol., 36: 161-183) which belongs to the same family of proteins as the p-glycoprotein pump P-gp (Broxterman, H. J., Giaccone, G., and Lankelma, J. (1995), Current Opinion in Oncology, 7:532-540) but shares less than 15% homology of amino acids with P-gp (Komorov, P. G., Shtil, A. A., Holian, O., Tee, L., Buckingham, L., Mechetner, E. B., Roninson, I. B., and Coon, J. S. (1998), Oncology Research, 10: 185-192). MRP has been found to occur naturally in a number of normal tissues, including liver, adrenal, testis, and peripheral blood mononuclear cells (Krishan, A., Fitz, C. M., and Andritsch, I. (1997), Cytometry 29: 279-285). MRP has also been identified in tissues of the lung, kidney, colon, thyroid, urinary bladder, stomach, spleen (Sugawara, I. (1998) The Cancer Journal 8(2)) and skeletal muscle (Kruh, G. D., Gaughan, K. T., Godwin, A., and Chan, A. (1995) Journal of the National Cancer Institute 87(16): 1256-1258). High levels of MRP have been implicated in multidrug resistance (MDR) in cancers of the lung and pancreas (Miller, D. W., Fontain, M., Kolar, C., and Lawson, T. (1996) Cancer Letters 107: 301-306), and in neuroblastomas, leukemias and cancer of the thyroid (Kruh, G. D., Gaughan, K. T., Godwin, A., and Chan, A. (1995) Journal of the National Cancer Institute 87(16): 1256-1258), as well as bladder, ovarian and breast cancers (Barrand, M., Bagrij, T., and Neo, S. (1997) General Pharmacology 28(5): 639-645). MRP-mediated MDR involves some of the same classes of compounds as those which are mediated by P-gp, including vinca alkaloids, epipodophyllotoxins, anthracyclins and actinomycin D (Barrand, M., Bagrij, T., and Neo, S. (1997) General Pharmacology 28(5): 639-645). However, the substrate specificity has been demonstrated to differ from that of P-gp (Komorov, P. G., Shtil, A. A., Holian, O., Tee, L., Buckingham, L., Mechetner, E. B., Roninson, I. B., and Coon, J. S. (1998) Oncology Research 10: 185-192). Drugs which would inhibit or which are not substrates for the MDR pump would, therefore, be useful as chemotherapeutic agents.
Some cancer cell lines, which have been induced to develop resistance to one type of microtubule interactive agent such as Taxol, have been found to be sensitive to other types of microtubule agents. For example, the chemically unrelated compounds epothilones (A and B), which are isolated from a myxobacterium, Sorangium cellulosum and are composed of 16 membered macrolides (Bollag, D. M., P. A. McQueney, J. Zhu et al. (1995) Cancer Res. 55:2325-2333) enhance microtubule stability, block cells in the G2/M phase of the cell cycle and prevent microtubule depolymerization in cancer cells, similar to Taxol. The epothilones also have a much greater cytotoxicity against p-glycoprotein expressing, multidrug resistant cells compared to non-multi-drug resistant cell lines.
Laulimalide and isolaulimalide, are two compounds which share Taxol""s microtubule-stabilizing activity (Mooberry, S. L., G. Tien, A. H. Hernandez et al. (1999) Cancer Res. 59:653-660), but are not chemically related to Taxol. Laulimalide is a potent inhibitor of cellular proliferation with IC50 values in the low nanomolar range, whereas isolaulimalide is much less potent with IC50 values in the low micromolar range. Both compounds inhibit cellular replication at the G2/M phase of the cell cycle. Laulimalide and isolaulimalide inhibit the proliferation of SKVLB-1 cells, a Pgp overexpressing multidrug-resistant cell line, again, suggesting that they are poor substrates for transport by Pgp.
Discodermolide, a compound derived from the marine sponge, Discodermia dissoluta (Gunasekera, S. P., M. Gunasekera, R. E. Longley (1990) J. Org. Chem. 55:4912-4915), is a potent inhibitor of cellular proliferation and has a similar mechanism of action to Taxol. Discodermolide blocks cells in the G2/M phase of the cell cycle (Longley, R. E., S. P. Gunasekera, D. Faherty et al. (1993) Immunosuppression by discodermolide. In: A. C. Allison ed. Annals of the New York Academy of Sciences Conference Proceedings, xe2x80x9cImmunosuppressive and Anti-inflammatory Drugsxe2x80x9d Vol. 696, April 12-15) and induces the hyperstabilization of microtubules in cells, leading to cell death (ter Harr, E., Kowalski et al. (1996) Biochemistry 35:243-250). Discodermolide also inhibits the proliferation of Pgp overexpressing, multidrug-resistant cell lines (Kowalski, R. J. et al. (1997) Mol. Pharmacol. 52:613-622).
The prevention and control of inflammation is also of great importance for the treatment of humans and animals. Much research has been devoted to development of compounds having anti-inflammatory properties. Certain methods and chemical compositions have been developed which aid in inhibiting or controlling inflammation, but additional anti-inflammatory methods and compositions are needed.
Immunomodulation is a developing segment of immunopharmacology. Immunomodulator compounds and compositions, as the name implies, are useful for modulating or regulating immunological functions in animals. Immunomodulators may be immunostimulants for building up immunities to, or initiate healing from, certain diseases and disorders. Conversely, immunomodulators may be immunoinhibitors or immunosuppressors for preventing undesirable immune reactions of the body to foreign materials, or to prevent or ameliorate autoimmune reactions or diseases.
Immunomodulators have been found to be useful for treating systemic autoimmune diseases, such as lupus erythematosus and diabetes, as well as immunodeficiency diseases. Further, immunomodulators may be useful for immunotherapy of cancer or to prevent rejections of foreign organs or other tissues in transplants, e.g., kidney, heart, or bone marrow.
Various immunomodulator compounds have been discovered, including FK506, muramylic acid dipeptide derivatives, levamisole, niridazole, oxysuran, flagyl, and others from the groups of interferons, interleukins, leukotrienes, corticosteroids, and cyclosporins. Many of these compounds have been found, however, to have undesirable side effects and/or high toxicity. New immunomodulator compounds are therefore needed to provide a wider range of immunomodulator function.
Dictyostatin-1 is a macrolide of polyketide origin which was first reported by Pettit et al. from a sponge of the genus Spongia collected in the Republic of the Maldives. (G. R. Pettit, Z. A. Cichacz, F. Gao, M. R. Boyd and J. M. Schmidt, (1994), xe2x80x9cIsolation and Structure of the cancer Cell Growth hibitor Dictyostatin-1xe2x80x9d J. Chem. Soc. Chem. Comm. 1111-1112) U.S. Pat. No. 5,430,053 (incorporated herein in its entirety by reference) describes the isolation and structure of dictyostatin-1 as well as its ability to inhibit the growth of various cancer cell lines in vitro. These reports do not disclose any utility for the compound against multi-drug resistant tumors in animals or humans or the ability of dictyostatin-1 to induce microtubule hyperstabilizing activity.
The subject invention provides novel uses for the dictyostatin class of compounds. These new uses include the control of cellular proliferation, cytotoxicity against human tumor cells resistant to chemotherapeutic agents, immunomodulation, and the control of inflammation. These uses arise from the identification of the role of dictyostatin compounds as tubulin polymerizers and microtubule stabilizers.
In accordance with the subject invention it has been determined that, when tubulin is treated with dictyostatin-1, a rapid onset of polymerization occurs in the absence of cells. This effect is not reversed upon temperature change indicating a long term stabilization of the microtubules. Also, PANC-1 human pancreatic adenoma cells treated with dictyostatin-1 do not undergo mitosis and show pronounced rearrangement of the microtubules in the cells.
The identification of dictyostatins as tubulin polymerizers and microtubule stabilizers facilitates the use of these compounds in a variety of clinical settings. The ability of the compound to inhibit the proliferation of human tumor cells which possess resistance to various chemotherapeutic agents is also embodied in this invention. The subject invention, would, therefore, have utility in the treatment of various human cancers which may have developed resistance to certain chemotherapeutic agents. Thus, the compounds of the subject invention are useful in the treatment of multi-drug resistant cancers.
The effects of dictyostatin-1 on tubulin polymerization microtubule stabilization and cytotoxicity towards human tumor cells which possess resistance to various chemotherapeutic agents could not be predicted either from the structure of the dictyostatin compounds or the previously reported data. In view of the unique and advantageous mode of action of the dictyostatin compounds, these compounds can be used in the treatment of a number of conditions in which aberrant cellular proliferation occurs. These conditions include, for example, autoimmune disorders and inflammatory diseases. In addition to use in the treatment of these disorders as well as other conditions involving pathological cellular proliferation, the compounds of the current invention can also be used as biochemical tools to study the process of tubulin polymerization/depolymerization and drug resistance.
In a preferred embodiment, the dictyostatin-1 compound of the subject invention has the following structure: 
Specifically exemplified herein is the use of dictyostatin-1 and/or salts, analogs, and derivatives thereof, for immunomodulation, control of inflammation, inhibiting cellular proliferation, stabilization of microtubules, induction of polymerization of tubulin and/or inhibiting cellular proliferation of multi-drug resistant cells.
A further aspect of the subject invention is an efficient process of obtaining dictyostatins from the lithistid sponge of the family Corallistidae.